Cell Free Biosynthesis of High-Quality Nucleic Acid and Uses Thereof

Abstract

The invention provides an improved cell free amplification method capable of producing large quantities of therapeutic-quality nucleic acids and methods of using the synthesized nucleic acid in research, therapeutic and other applications—The methods combine several different state-of-the-art procedures and coordinate their applications to affordably synthesize nucleic acids for therapeutic purposes. It combines in vitro rolling circle amplification, high fidelity polymerases, high affinity primers, and streamlined template specifically designed for particular applications. For expression purposes, the templates contain an expression cassette including a eukaryotic promoter, the coding sequence for the gene of interest, and a eukaryotic termination sequence. Following amplification, concatamers are subsequently processed according to their intended use and may include: restriction enzyme digestion for the production of short expression cassettes (SECs); ligation steps to circularize the SEC (CNAs); and/or supercoiling steps to produce sCNAs. The final product contains nearly non-detectable levels of bacterial endotoxin.

1 . A process for producing high-quality nucleic acid in a cell free system, comprising:
 (a) combining a circular template in a reaction mixture with one or more primers, which are complementary to at least one strand of the circular template, to form a template-primer complex;   (b) incubating the template-primer complex with at least one high-fidelity nucleic acid polymerase to produce a concatamer comprising tandem units of the circular template; and   (c) cutting the concatamer into smaller fragments comprising at least one delivery unit having a sequence of interest.   
     
     
         2 . The process according to  claim 1 , further comprising:
 processing the smaller fragments by the following steps:   filling in or removing the ends of the smaller fragments;   ligating ends of the smaller fragments to produce circularized smaller fragments; and   supercoiling the circularized smaller fragments.   
     
     
         3 . The process according to  claim 2 , the circularized smaller fragments are modified with one or more of the following modifications:
 (i) modification at one end with a modified base, a fluorescent tag, a magnetic tag, a radiolabeled tag, or a bio-physical tag;   (ii) modification at an internal base by adding modified bases at step (b);   (iii) modification by adding one or more modified primers at step (a) or (b); and   (iv) conjugation with a nucleic acid, a protein, a peptide, a polymer, or a small molecule.   
     
     
         4 . The process according to  claim 1 , the smaller fragments are modified with one or more of the following modifications:
 (i) modification at one end with a modified base a fluorescent tag a magnetic tag, a radiolabeled tag, or a bio-physical tag;   (ii) modification at an internal base by adding modified bases at step (b);   (iii) modification by adding one or more modified primers at step (a) or (b); and   (iv) conjugation with a nucleic acid a protein, a peptide a polymer, or a small molecule.   
     
     
         5 . The process according to  claim 1 , wherein the delivery unit comprises one or more expression cassettes. 
     
     
         6 . The process according to  claim 1 , wherein the high-fidelity nucleic acid polymerase is at least one selected from the group consisting of Phi29 DNA polymerase, Phi29-like DNA polymerase, M2 DNA polymerase, B103 DNA polymerase, GA-1 DNA polymerase, phi-PRD1 polymerase, VENT DNA polymerase, DEEP VENT DNA polymerase, KlenTaq DNA polymerase, DNA polymerase I, Klenow fragment of DNA polymerase I, DNA polymerase III, T3 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Bst polymerase, rBST DNA polymerase, N29 DNA polymerase, TopoTaq DNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase and a derivative thereof. 
     
     
         7 . (canceled) 
     
     
         8 . The process according to  claim 1 , wherein the cutting of the concatamer is accomplished by using a restriction enzyme. 
     
     
         9 . A research, diagnostic, or therapeutic composition comprising the delivery unit prepared by the process according to  claim 1 . 
     
     
         10 . A method for preventing or treating a disease or genetic disorder of a human, animal or plant, comprising: administering an effective amount of the composition of  claim 9  to a subject in need thereof. 
     
     
         11 . The method according to  claim 10 , wherein the disease is caused by a virus selected from HIV, influenza virus, parainfluenza virus, adenovirus, corona virus, herpes simplex virus, herpes zoster virus, papilloma virus, and rhino virus. 
     
     
         12 . The method according to  claim 10 , wherein the disease is caused by bacteria, mycobacteria, eubacteria or fungi. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 10 , wherein the composition is delivered by injection, an aerosol, an oral composition, eyedrops, suppositories, topical ointments, skin patches and soaks, surgically implanted devices, electroporation, pressure-mediated or hydrodynamic injection, ultrasonic nebulization, particle bombardment, or a long-term release system. 
     
     
         15 . An apparatus for producing high-quality nucleic acid, comprising:
 a reaction vessel having at least one entry port and one exit port that before addition of reaction reagents, is clean, sterile and free of contaminating nucleic acid sequences;   an input device attached to the entry port and which feeds at least one outside component from an outside holding chamber to the reaction vessel;   at least one outside holding chamber connected to the input device;   a means for pumping an outside component from the holding chamber, through the input device, into the reaction vessel;   an export device attached to the exit port of the reaction vessel;   at least one outside receiving chamber connected to the export device;   a means for regulating temperature of the reaction vessel;   a means for monitoring and controlling the progress of a reaction mixture within the reaction vessel; and   a means for mixing the reaction mixture.   
     
     
         16 . The apparatus of  claim 15  wherein the reaction vessel is made of either a flexible material wherein the means for mixing is applied to the outside of the vessel or a hardened pre-formed material wherein the means for mixing is applied inside the vessel. 
     
     
         17 . The apparatus of  claims 15  further comprising a programmable computer to automate or remotely monitor, adjust or control one or more of the following: reaction viscosity, nucleic acid concentration, solution turbidity, conductivity, pH, temperature, protein content, endotoxin, bioburden, chemical contaminants, temperature, timing, addition of reagents, mixing, and dispensing of the reaction mixture. 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . The process of  claim 1 , wherein the circular template is devoid of one or more of: a plasmid replication sequence, a selection sequence, or a marker sequence 
     
     
         22 . The process of  claim 1 , further comprising:
 purifying the smaller fragments to meet one or more of the following criteria: level of genomic DNA is <10 ng (based on a human dose), level of RNA is non-detectable on 0.8% agarose gel (based on a human dose), level of bacterial protein is <10 ng (based on a human dose), level of bacterial endotoxin is <1 Unit/kg body weight or <0.1 EU/μg DNA, or is sterile.   
     
     
         23 . A method for inducing immunity in a human or an animal, comprising immunizing the human or animal with a composition comprising the smaller fragments prepared by the process of  claim 1 . 
     
     
         24 . The process of  claim 1 , further comprising addition of one or more of the following proteins: MutS, a single-stranded binding protein, an additional polymerase. 
     
     
         25 . The composition of  claim 9 , wherein the smaller fragments comprise one or more sequences derived from B-galactosidase, luciferase, HIV gp160 envelope protein, hepatitis B small surface antigen, influenza hemaglutinin, influenza neuraminidase, or a cytokine.